A carboxy-terminally truncated form of the human immunodeficiency virus type 1 Vpr protein induces apoptosis via G(1) cell cycle arrest

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carbo xy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce a poptosis via G(1) arrest of the cell cycle. Immunostaining for various mark ers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C8 1 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidyl serine on the plasma membrane, as an early indicator of apoptosis and measu rement of the activity of caspase-3, a signaling molecule in apoptotic path ways, indicated that C81 is a strong inducer of apoptosis. Expression of C8 1 induced the condensation, fragmentation, and clumping of chromatin that a re typical of apoptosis, Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-posi tive cells and the activity of caspase-3. Replacement of lie or Leu residue s by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like dom ain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1), arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized fo r G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, an d Ile74 also play an important role in the C81-induced suppression of growt h. These results suggest that the suppression of growth induced by C81 resu lt in apoptosis that is independent of G(2) arrest of the cell cycle.